EP1740693B1 - Vorrichtung zur kontinuierlichen kultivierung mit mobilem gefäss zur möglichen selektion von filterzellvarianten - Google Patents

Vorrichtung zur kontinuierlichen kultivierung mit mobilem gefäss zur möglichen selektion von filterzellvarianten Download PDF

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EP1740693B1
EP1740693B1 EP05723500A EP05723500A EP1740693B1 EP 1740693 B1 EP1740693 B1 EP 1740693B1 EP 05723500 A EP05723500 A EP 05723500A EP 05723500 A EP05723500 A EP 05723500A EP 1740693 B1 EP1740693 B1 EP 1740693B1
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tubing
culture
growth chamber
growth
medium
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EP1740693A1 (de
EP1740693A4 (de
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Eudes Francois Marie De Crecy
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/06—Tubular
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/26—Constructional details, e.g. recesses, hinges flexible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements

Definitions

  • the described invention provides a method and a device that allow selection of living cells, with increased rates of reproduction and specific metabolic properties, in a liquid or semi-solid medium.
  • selection adaptive evolution
  • genetically variant organisms arise in a population and compete with other variants of the same origin.
  • Those with the fastest rate of reproduction increase in relative proportion over time, leading to a population (and individual organisms) with increased reproductive rate.
  • This process can improve the performance of organisms used in industrial processes or academic purpose.
  • Serial culture involves repetitive transfer of a small volume of grown culture to a much larger vessel containing fresh growth medium. When the cultured organisms have grown to saturation in the new vessel, the process is repeated. This method has been used to achieve the longest demonstrations of sustained culture in the literature ( Lenski & Travisano: Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations. 1994. Proc Natl Acad Sci USA. 15:6808-14 ), in experiments which clearly demonstrated consistent improvement in reproductive rate over period of years. This process is usually done manually, with considerable labor investment, and is subject to contamination through exposure to the outside environment. Serial culture is also inefficient, as described in the following paragraph.
  • the rate of selection is dependant on population size ( Fisher: The Genetical Theory of Natural Selection.1930. Oxford University Press, London, UK ). Furthermore, in a situation like serial transfer where population size fluctuates rapidly, selection is proportional to the harmonic mean ( ⁇ ) of the population ( Wright: Size of population and breeding structure in relation to evolution. 1938. Science 87: 430-431 ), and hence can be approximated by the lowest population during the cycle.
  • Continuous culture as distinguished from serial dilution, involves smaller relative volume such that a small portion of a growing culture is regularly replaced by an equal volume of fresh growth medium. This process maximizes the effective population size by increasing its minimum size during cyclical dilution.
  • Devices allowing continuous culture are termed “chemostats” if dilutions occur at specified time intervals, and “turbidostats” if dilution occur automatically when the culture grows to a specific density.
  • Chemostats were invented simultaneously by two groups in the 1950's ( Novick & Szilard: Description of the chemostat. 1950. Science 112: 715-716 ) and ( Monod: La technique de la culture continue - Théorie et applications.1950. Ann. Inst. Pasteur 79:390-410 ). Chemostats have been used to demonstrate short periods of rapid improvement in reproductive rate ( Dykhuizen DE. Chemostats used for studying natural selection and adaptive evolution.1993. Methods Enzymol. 224:613-31 ).
  • This method and device achieves the goal, but requires independent complex manipulations of several fluids within a sterile (sealed) environment, including one (NaOH) which is both very caustic and potentially very reactive, quickly damaging valves, and posing containment and waste-disposal problems.
  • JP 03,030,665 describes an automatic subculturing device for microscopic organisms or cells comprising a flexible tube where culture medium is filled and many clamps valves that can tighten or loosen said tube and which are shifted along it to create a new growth chamber.
  • US 6,066,497 describes a cell apparatus comprising, mounted on a rotor, a peristaltic pump with channels which are linked to bottles containing a cell culture, the pump being able to empty the bottles thanks to pump rollers.
  • the channels do not move.
  • the device provides a means for regular dilution of a grown culture with fresh growth medium, a means for gas exchange between the culture and the outside environment, sterility, and automatic operation as either a chemostat or a turbidostat.
  • the present invention is designed to achieve this goal without any fluid transfer, Including sterilization or rinsing functions. This represents a specific advantage of the present invention with respect to prior art in so far as It avoids the hazards and difficulties associated with sterilization and rinsing, including containment and complex fluid transfers involving caustic solvents.
  • the medium and the chamber surface are static with respect to each other, and both are regularly and simultaneously replaced by peristaltic movement of the tubing through "gates", or points at which the tube Is sterilely subdivided by clamps that prevent the cultured organisms from moving between regions of the tube.
  • UV gates can also (optionally) be added upstream and downstream of the culture vessel for additional security.
  • the present method and device are also an Improvement over prior art insofar as they continually, rather than periodically, select against adherence of dilution-resistant variants to the chemostat surfaces, as replacement of the affected surfaces occurs in tandem with the process of dilution.
  • the tube is subdivided in a transient way such that there are regions that contain saturated (fully grown) culture, regions that contain fresh medium, and a region between these two, termed the growth chamber, in which grown culture is mixed with fresh medium to achieve dilution.
  • the gates are periodically released from one point on the tube and replaced at another point, such that grown culture along with Its associated growth chamber surface and attached static organisms, is removed by isolation from the growth chamber and replaced by both fresh medium and fresh chamber surface.
  • static variants are specifically counter-selected by removal from the region in which selection is occurring (the growth chamber).
  • FIG. 1 One potential configuration for the present device is shown in figure 1 , as it appears after having been loaded with a fresh tube of sterile medium (shown divided into regions A-H by said gates (3), (4) and (5)).
  • Inoculation of the device with the chosen organism could be achieved by introduction of the organism into the growth chamber ( fig 3 ), through injection ( figure 4 , region B). The culture would then be allowed to grow to the desired density and continuous culture would begin ( fig 5 ).
  • Continuous culture would proceed by repetitive movements of the gated regions of tubing. This involves simultaneous movements of the gates, the tubing, the medium, and any culture within the tubing.
  • the tubing will always move in the same direction; unused tubing containing fresh medium (and hereafter said to be 'upstream' of the growth chamber (7)) will move into the growth chamber and mix with the culture remaining there, providing the substrate for further growth of the organisms contained therein.
  • this medium and its associated tubing Before introduction into the growth chamber region, this medium and its associated tubing will be maintained in a sterile condition by separation from the growth chamber by the upstream gates (3). Used tubing containing grown culture will simultaneously be moved 'downstream' and separated from the growth chamber by the downstream gates (4).
  • Gate configuration is not a specific point of the present patent application.
  • gates can be designed through one chain of multiple teeth simultaneously moved or in another configuration separated in distinct synchronized chains as depicted in figure 1 .
  • Gates can consist of a system made of two teeth pinching the tubing in a stacking manner as described in figure 10 , avoiding contamination between regions G and H of the tubing through the precision of the interface between the teeth.
  • sterile gates can be obtained by pressing one tooth against one side of the tubing and thereby pressing the tubing tightly against a fixed chassis along which tubing is slid during its peristaltic movement, as sketched in figure 3 to 9 , marks 3, 4 and 5.
  • Said thermostatically controlled box (2) is obtained by already known means such as a thermometer coupled with a heating and cooling device.
  • Aeration when required for growth of the cultured organism or by the design of the experiment, is achieved directly and without mechanical assistance by the use of gas permeable tubing.
  • flexible gas permeable tubing can be made of silicone. Aeration could be achieved through exchange with the ambient atmosphere or through exchange with an artificially defined atmosphere (liquid or gas) that contacts the growth chamber or the entire chemostat.
  • the flexible tubing can be gas impermeable.
  • flexible gas impermeable tubing can be made of coated or treated silicone.
  • regions of the tubing can also be confined in a specific and controlled atmospheric area to control gas exchange dynamics. This can be achieved either by making said thermostatically controlled box gastight and then injecting neutral gas into it or by placing the complete device in an atmosphere controlled room.
  • the device is further designed to be operable in a variety of orientations with respect to gravity, that is, to be tilted as shown by figure 2 , along a range of up to 360°.
  • Dilution-resistant variants may avoid dilution by sticking to one another, rather than to the chamber wall if aggregated cells can fall upstream and thereby avoid removal from the chamber.
  • the tubing generally be tilted downward, such that aggregated cells will fall toward the region that will be removed from the growth chamber during a cycle of tube movement. This configuration involves tilting the device so that the downstream gates are below the upstream gates with respect to gravity.
  • the growing chamber can be depressurized or over pressurized according to conditions chosen by the experimenter. Different ways of adjusting pressure can be used, For instance applying vacuum or pressurized air to the fresh medium and tubing through its upstream extremity and across the growth chamber; another way of depressurizing or overpressurizing tubing can be done by alternate pinching and locking tubing upstream of the growth chamber.
  • Tilting movements of the device, and/or shaking of the growth chamber by an external device (9) can be used to decrease aggregation of cells within the growth chamber.
  • one or several stirring bars can be included in the tubing filled with fresh medium before sterilization and magnetically agitated during culture operations.
  • the proportional length of the regions of fresh medium defined by the upstream gates as compared to the length of the culture chamber will define the degree of dilution achieved during a cycle.
  • the frequency of dilution can be determined either by timing (chemostat function) or by feedback regulation whereby the density of the culture in the growth chamber is measured by a turbidimeter ( figure 1 - mark 6) and the dilution cycle occurs when the turbidity reaches a threshold value (turbidostat function).
  • the sampling chamber allows withdrawing grown culture in order to analyze the outcome of an experiment, collect organisms with improved growth rate for further culture, storage, or functional implementation, or other purposes such as counting the population, checking the chemical composition of the medium, or testing the pH of grown culture.
  • tubing can include by construction a pH indicator line embedded/encrusted in the wall of the tubing.
  • Any form of liquid or semi-solid material can be used as a growth medium in the present device.
  • the ability to utilize semi-solid growth substrates is a notable advancement over prior art.
  • the growth medium which will define the metabolic processes improved by the selection process, can be chosen and defined by the user.
  • this device can contain multiple growth chambers, such that the downstream gates of one growth chamber become the upstream gates of another. This could, for example, allow one organism to grow alone in the first chamber, and then act as the source of nutrition for a second organism (or virus) in the second chamber.
  • This device and method allows researchers and product developers to evolve any strain of culturable living cells in suspension through sustained growth (continuous culture); the resulting improved organism can constitute a new strain or species. These new organisms can be identified by mutations acquired during the course of culture, and these mutations may allow the new organisms to be distinguished from their ancestors genotype characteristics. This device and method allow the researcher to select new strains of any living organism by segregating individuals with improved rates of reproduction through the process of natural selection.

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Claims (22)

  1. Vorrichtung zum Steigern der Reproduktionsrate (durch gesteigerte Reproduktionsgeschwindigkeit und/oder gesteigerte Reproduktionsausbeute) lebender Zellen in Suspension oder anderer kultivierbarer Organismen, insbesondere Bakterien, Archaeen, Eukaryoten und Viren, durch ein natürliches Selektionsverfahren, wobei die Vorrichtung aufweist:
    a) einen flexiblen, sterilen Schlauch (1) enthaltend Kulturmedium,
    b) ein System von Toren (3, 4, 5) realisiert durch Klemmen, welche den Schlauch unterteilen in:
    - einen Stromaufwärtsbereich, enthaltend frisches Wachstumsmedium (7),
    - eine Wachstumskammer (10), enthaltend wachsende Kultur und angeordnet zwischen Stromaufwärts- und Stromabwärtsbereichen,
    - und eine Stromabwärtsregion, enthaltend erschöpfte Kultur (15),
    c) ein Mittel zum Bewegen des Schlauches (1) gegenüber den Toren (3, 4, 5), so daß ein Teil (B) der Wachstumskammer und die zugehörige Kultur abgeklemmt und von der Wachstumskammer getrennt werden kann und so daß ein Teil (D) eines frischen, unbenutztes Medium enthaltenden Schlauchs, mit einem Teil (C) der Kultur und entsprechendem Medium, welche bereits in der Wachstumskammer vorhanden sind, verbunden werden kann,
    dadurch gekennzeichnet, daß der Schlauch (1), das Medium und die Kultur im Schlauch durch gleichzeitige peristaltische Bewegungen der Tore beweglich sind.
  2. Vorrichtung nach Anspruch 1, wobei das System von Toren weiter den Schlauch (1) in eine Probengewinnungskammer (11) unterteilt, welche stromabwärts der Wachstumskammer angeordnet ist.
  3. Vorrichtung nach Anspruch 1 oder 2, wobei das System von Toren weiter den Schlauch (1) in mehrere Wachstumskammern unterteilt, wobei das stromabwärts gelegene Tor einer Wachstumskammer das stromaufwärts gelegene Tor einer anderen wird.
  4. Vorrichtung nach einem der Ansprüche 1 bis 3, wobei der Schlauch flexibel ist, um das Klemmen und das Aufteilen in getrennte Kammern zu ermöglichen.
  5. Vorrichtung nach einem der Ansprüche 1 bis 4, wobei die Tore durch eine oder mehrere Ketten mehrerer simultan bewegter Zähne ausgebildet sind.
  6. Vorrichtung nach einem der Ansprüche 1 bis 5, wobei die Tore aus einem System bestehen, das aus zwei den Schlauch in geschichteter Weise quetschenden Zähne aufgebaut ist, wobei durch die Genauigkeit der Kontaktfläche zwischen den Zähnen eine Kontamination zwischen Abschnitten (G, H) des Schlauches vermieden ist.
  7. Vorrichtung nach einem der Ansprüche 1 bis 6, wobei der Schlauch gasdurchlässig ist, beispielsweise indem er vorwiegend aus Silikon besteht, um je nach Art des Experimentes einen Gasaustausch zwischen den gezüchteten Organismen und der Außenwelt zu ermöglichen.
  8. Vorrichtung nach einem der Ansprüche 1 bis 6, wobei der Schlauch gasundurchlässig ist, um im Falle eines Anaerobiose verlangenden Experimentes einen Gasaustausch zwischen dem Schlauch und der Außenwelt zu verhindern.
  9. Vorrichtung nach einem der Ansprüche 1 bis 8, wobei der Schlauch, um Trübungsmessungen zu ermöglichen, transparent oder transluzent ist.
  10. Vorrichtung nach Anspruch 9, weiter aufweisend einen Trübungsmesser (6) und ein Steuersystem (13), um die optische Dichte der wachsenden Kultur zu überwachen und eine kontrollierte Steuerung des Schlauches (1) auf Basis der Kulturdichte zu bewirken.
  11. Vorrichtung nach einem der Ansprüche 1 bis 10, weiter aufweisend ein Mittel zum, bezogen auf die Umgebungsatmosphäre, Druck mindern oder mit Druck beaufschlagen des Wachstumskammerschlauches und zugehörigen Medien und Kulturen je nach experimentellen Anforderungen.
  12. Vorrichtung nach einem der Ansprüche 1 bis 11, weiter aufweisend eine pH-Indikator-Linie eingebettet in/überziehend die Wand des Schlauches um eine permanente Überwachung des pH-Wertes in der Wachstumskammer zu erreichen.
  13. Vorrichtung nach einem der Ansprüche 1 bis 12, weiter aufweisend ein Mittel (2) zum Heizen oder Kühlen des Wachstumskammerschlauches und zugehöriger Medien und Kulturen entsprechend experimentellen Bedingungen.
  14. Vorrichtung nach einem der Ansprüche 1 bis 13, weiter aufweisend ein Mittel (9) zum Bewegen oder ruhig halten des Wachstumskammerschlauches und zugehöriger Medien und Kulturen.
  15. Vorrichtung nach Anspruch 14, wobei der Schlauch für Durchmischungszwecke ein oder mehrere Rührstäbchen haben kann.
  16. Vorrichtung nach einem der Ansprüche 1 bis 15, weiter aufweisend ein Mittel (2) zum Einschließen von Bereichen des Schlauches in einem bestimmten und kontrollierten Atmosphärenbereich, um eine Gasaustauschdynamik zu steuern.
  17. Vorrichtung nach den Ansprüchen 1 bis 16, weiter aufweisend ein Mittel (2) zum Kippen des Wachstumskammerschlauches und zugehöriger Medien und Kulturen entweder nach unten, um angesammelte Zellen zu entfernen, oder nach oben, um Luft zu entfernen.
  18. Verfahren, welches die Reproduktionsrate lebender Zellen in Suspension oder anderer kultivierbarer Organismen, insbesondere Bakterien, Aracheen, Eukaryoten und Viren, durch einen natürlichen Selektionsprozeß steigert, wobei das Verfahren aufweist:
    a) Umverteilen eines flexiblen, frisches Wachstumsmedium (7) enthaltenden Schlauches (1) durch Verschließen von Toren (3, 4, 5) in einen Stromaufwärtsbereich (7), enthaltend frisches Wachstummedium, eine Wachstumskammer (10), enthaltend Wachstumskulturen und angeordnet zwischen Stromaufwärts- und Stromabwärtsbereichen, eine Probengewinnungskammer (11) angeordnet zwischen der Wachstumskammer, und einen Stromaufwärtsbereich (15), enthaltend verbrauchte Kulturen,
    b) Bereitstellen einer Anfangskultur in der beschriebenen Wachstumskammer (10) durch sterile Injektion einer Startkultur,
    c) Beibehalten von Wachstumsbedingungen entsprechend experimentellen Anforderungen,
    d) nach bestimmtem Wachstum der Kulturen Anwenden gleichzeitiger peristaltischer Bewegungen der Tore (3, 4, 5) zum Bewegen des Schlauches (1) und des Mediums und der Kulturen im Schlauch, um gleiche Teile von frischem Medium bzw. Wachstumskulturen in bzw. aus dem als Wachstumskammer festgelegten Bereich zu bewegen, Ermöglichen, daß sich verbleibende Teile der gewachsenen Kulturen mit eingeführten Teilen frischen Mediums mischen und weiterwachsen,
    e) Wiederholen der Schritte c) und d) bis zum Ende des Experimentes, um gleichmäßige Kulturen und eine Selektion von Varianten mit gesteigerter Reproduktionsrate zu erreichen,
    f) anforderungsgemäßes Abziehen einer Probe gewachsener Kulturen aus der Probengewinnungskammer (11).
  19. Verfahren gemäß Anspruch 18, wobei Schritt c) umfaßt das Beibehalten der Wachstumsbedingungen entsprechend Temperatur, Druck, optische Dichte, chemischer Aktivität, Durchmischen und/oder Begasen mit verschiedenen Gasen.
  20. Verfahren nach Anspruch 18 oder 19, wobei Schritt c) weiter zum Steuern einer Gasaustauschdynamik das Einschließen von Bereichen des Schlauches in einem bestimmten und kontrollierten Atmosphärenbereich umfaßt.
  21. Verfahren nach einem der Ansprüche 18 bis 20, wobei Schritt c) weiter eine Kombination aus Kippen der Vorrichtung und Betätigen von Mischern führend zu einem geeigneten Mischen zum Durchmischen der gewachsenen Kulturen umfaßt, um eine Aggregation lebender Organismen zu verhindern oder zu unterdrücken.
  22. Verfahren nach einem der Ansprüche 18 bis 21, wobei Schritt a) weiter das Unterteilen des Schlauches in mehrere Wachstumskammern umfaßt, wobei das stromabwärts gelegene Tor einer Wachstumskammer das stromaufwärts gelegene Tor einer anderen Wachstumskammer wird, und wobei Schritt b) weiter das Bereitstellen eines zweiten Organismus in der anderen Wachstumskammer umfaßt.
EP05723500A 2004-02-23 2005-02-23 Vorrichtung zur kontinuierlichen kultivierung mit mobilem gefäss zur möglichen selektion von filterzellvarianten Active EP1740693B1 (de)

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US (1) US7939315B2 (de)
EP (1) EP1740693B1 (de)
JP (1) JP4771433B2 (de)
CN (1) CN1973028B (de)
AU (2) AU2005217618C1 (de)
BR (1) BRPI0507983B1 (de)
CA (1) CA2557574C (de)
DK (1) DK1740693T3 (de)
ES (1) ES2389412T3 (de)
IL (1) IL177608A (de)
MX (1) MXPA06009596A (de)
NO (1) NO336517B1 (de)
NZ (1) NZ591884A (de)
PL (1) PL1740693T3 (de)
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SG (1) SG169364A1 (de)
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US20070037276A1 (en) * 2004-02-23 2007-02-15 Eudes Francois Marie De Crecy Continuous culture apparatus with mobile vessel, allowing selection of fitter cell variants and producing a culture in a continuous manner
CN101668843A (zh) * 2007-04-27 2010-03-10 东洋制罐株式会社 细胞培养装置、细胞培养体系及细胞培养方法
FR2927906B1 (fr) * 2008-02-21 2010-04-02 Eco Solution Procede et dispositif de culture cellulaire en mode continu ouvert.
US20110306101A1 (en) * 2008-07-11 2011-12-15 De Crecy Eudes method of producing fatty acids for biofuel, biodiesel, and other valuable chemicals
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US7939315B2 (en) 2011-05-10
RU2373273C2 (ru) 2009-11-20
NO20064286L (no) 2006-11-14
AU2010257471B2 (en) 2011-09-29
ES2389412T3 (es) 2012-10-26
PL1740693T3 (pl) 2012-11-30
CA2557574C (en) 2013-04-30
NO336517B1 (no) 2015-09-14
IL177608A0 (en) 2006-12-10
JP4771433B2 (ja) 2011-09-14
CN1973028B (zh) 2013-09-18
US20080220501A1 (en) 2008-09-11
AU2005217618C1 (en) 2011-09-01
CN1973028A (zh) 2007-05-30
EP1740693A1 (de) 2007-01-10
SG169364A1 (en) 2011-03-30
AU2005217618A1 (en) 2005-09-09
EP1740693A4 (de) 2007-02-14
NZ591884A (en) 2012-07-27
IL177608A (en) 2013-03-24
AU2010257471A1 (en) 2011-02-03
CA2557574A1 (en) 2005-09-09
BRPI0507983B1 (pt) 2015-04-22
ZA200607310B (en) 2009-01-28
RU2006133936A (ru) 2008-03-27
WO2005083052A1 (en) 2005-09-09
AU2005217618A2 (en) 2005-09-09
MXPA06009596A (es) 2007-03-26
AU2005217618B2 (en) 2011-01-27
WO2005083052B1 (en) 2005-10-27
JP2007522825A (ja) 2007-08-16
BRPI0507983A (pt) 2007-07-24

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